Streptococcus pneumoniae is an important cause of otitis media, meningitis, bacteremia and pneumonia, and a leading cause of fatal infections in the elderly and persons with underlying medical conditions, such as pulmonary disease, liver disease, alcoholism, sickle cell, cerebrospinal fluid leaks, acquired immune deficiency syndrome (AIDS), and patients undergoing immunosuppressive therapy. It is also a leading cause of morbidity in young children. Pneumococcal infections cause approximately 40,000 deaths in the U.S. each year (CDC. Prevention of Pneumococcal Disease. MMWR 1997; 46:1-25). The most severe pneumococcal infections involve invasive meningitis and bacteremia infections, of which there are 3,000 and 50,000 cases annually, respectively.
Despite the use of antibiotics and vaccines, the prevalence of pneumococcal infections has declined little over the last twenty-five years; the case-fatality rate for bacteremia is reported to be 15-20% in the general population, 30-40% in the elderly, and 36% in inner-city African Americans. Less severe forms of pneumococcal disease are pneumonia, of which there are 500,000 cases annually in the U.S., and otitis media in children, of which there are an estimated 7,000,000 cases annually in the U.S. caused by S. pneumoniae. Strains of drug-resistant S. pneumoniae are becoming ever more common in the U.S. and worldwide. In some areas, as many as 30% of pneumococcal isolates are resistant to penicillin. The increase in antimicrobial resistant pneumococcus further emphasizes the need for preventing pneumococcal infections.
Pneumococcus asymptomatically colonizes the upper respiratory tract of normal individuals; disease often results from the spread of organisms from the nasopharynx to other tissues during opportunistic events. The incidence of carriage in humans varies with age and circumstances. Carrier rates in children are typically higher than are those of adults. Studies have demonstrated that 38 to 60% of preschool children, 29 to 35% of grammar school children and 9 to 25% of junior high school children are carriers of pneumococcus. Among adults, the rate of carriage drops to 6% for those without children at home, and to 18 to 29% for those with children at home. It is not surprising that the higher rate of carriage in children than in adults parallels the incidence of pneumococcal disease in these populations.
An attractive goal for streptococcal vaccination is to reduce carriage in the vaccinated populations and subsequently reduce the incidence of pneumococcal disease. There is speculation that a reduction in pneumococcal carriage rates by vaccination could reduce the incidence of the disease in non-vaccinated individuals as well as vaccinated individuals. This “herd immunity” induced by vaccination against upper respiratory bacterial pathogens has been observed using the Haemophilus influenzae type b conjugate vaccines (Takala, A. K., et al., J. Infect. Dis. 1991; 164: 982-986; Takala, A. K., et al., Pediatr. Infect. Dis. J., 1993; 12: 593-599; Ward, J., et al., Vaccines, S. A. Plotkin and E. A. Mortimer, eds., 1994, pp. 337-386; Murphy, T. V., et al., J. Pediatr., 1993; 122: 517-523; and Mohle-Boetani, J. C., et al., Pediatr. Infect. Dis. J., 1993; 12: 589-593).
It is generally accepted that immunity to Streptococcus pneumoniae can be mediated by specific antibodies against the polysaccharide capsule of the pneumococcus. However, neonates and young children fail to make adequate immune response against most capsular polysaccharide antigens and can have repeated infections involving the same capsular serotype. One approach to immunizing infants against a number of encapsulated bacteria is to conjugate the capsular polysaccharide antigens to protein to make them immunogenic. This approach has been successful, for example, with Haemophilus influenzae b (see U.S. Pat. No. 4,496,538 to Gordon and U.S. Pat. No. 4,673,574 to Anderson).
However, there are over ninety known capsular serotypes of S. pneumoniae, of which twenty-three account for about 85-90% of the disease. For a pneumococcal polysaccharide-protein conjugate to be successful, the capsular types responsible for most pneumococcal infections would have to be made adequately immunogenic. This approach may be difficult, because the twenty-three polysaccharides included in the presently-available vaccine are not all optimally immunogenic, even in adults.
Protection mediated by anti-capsular polysaccharide antibody responses is restricted to the polysaccharide type. Different polysaccharide types differentially facilitate virulence in humans and other species. Pneumococcal vaccines have been developed by combining the 23 different capsular polysaccharides which are representative of the prevalent types of human pneumococcal disease. These 23 polysaccharide types have been used in a licensed pneumococcal vaccine since 1983 (D. S. Fedson, M. Musher, Vaccines, S. A. Plotkin and J. E. A. Montimer, eds., 1994, pp. 517-564). The licensed 23-valent polysaccharide vaccine has a reported efficacy of approximately 60% in preventing bacteremia caused by vaccine type pneumococci in healthy adults.
However, the efficacy of the vaccine has been controversial, and at times, the justification for the recommended use of the vaccine questioned. It has been speculated that the efficacy of this vaccine is negatively affected by having to combine 23 different antigens. Having a large number of antigens combined in a single formulation may negatively affect the antibody responses to individual types within this mixture because of antigenic competition. The efficacy is also affected by the fact that the 23 serotypes encompass all serological types associated with human infections and carriage.
An alternative approach for protecting children, and also the elderly, from pneumococcal infection would be to identify protein antigens that could elicit protective immune responses. Such proteins may serve as a vaccine by themselves, may be used in conjunction with successful polysaccharide-protein conjugates, or as carriers for polysaccharides.
Russell et al. have described an immunogenic, species-common protein from S. pnuemoniae designated pneumococcal fimbrial protein A. (J. Clin. Microbiol. 28: 2191-95 (1990)). This 37 kDa protein antigen is also described in U.S. Pat. No. 5,422,427, the teachings of which are hereby incorporated in their entirety herein by reference. The 37 kDa protein, which was previously referred to as pneumococcal fimbral protein A, has more recently been designated pneumococcal surface protein A (PsaA). For the purposes of the present application, references made to PsaA, pneumococcal surface protein A, pnuemococcal fimbral protein A, or the 37 kDa antigen, shall all be understood to refer to that certain protein antigen from S. pneumoniae characterized by Russell et al. (1990) and described in U.S. Pat. No. 5,422,427.
Immunoblot analysis studies with a monoclonal antibody to PsaA demonstrate that PsaA is common to all 23 pneumococcal vaccine serotypes (Russell et al., 1990). The gene encoding PsaA has been cloned and sequenced. (Sampson et al. (1994) “Cloning and nucleotide sequence analysis of psaA, the Streptococcus pneumoniae gene encoding a 37-kilodalton protein homologous to previously reported Streptococcus sp. adhesins” Infect. Immun. 62:319-324. Unfortunately, the strain from which the gene was cloned, R36A, is an unencapsulated strain of low virulence, and subsequent studies have revealed that it is not representative of psaA genes from serotypes of clinically relevant strains. For example, oligonucleotide primers based on the published sequence of psaA from R36A were unable to direct PCR amplification of the psaA gene from strain D39, a virulent capsular type 2 strain (Berry and Paton. Infect. Immun. 64: 5255-62, 1996).
The psaA gene has been cloned from encapsulated strain 6B, and is the subject of pending patent application Ser. No. 08/222,179, now abandoned. This gene is more representative of clinically relevant strains. This gene was initially cloned into pUC18 and subsequently inserted into an expression vector, pQE30 (Quiagen, Calif.) containing the T5 promoter. However, while E. coli host cells transformed with this construct and induced with IPTG did express recombinant PsaA, the recombinant cells were unstable and yields were low. This instability may be due to the toxicity of naturally lipidated recombinant proteins to E. coli host cells; and makes such expression systems of limited use in preparation of sufficient quantities of recombinant PsaA for use in immunological compositions.
In order to establish an infection, S. pneumoniae must first gain entry to the host through mucosal surfaces. The principal determinant of specific immunity at mucosal surfaces is secretory IgA (S-IgA) which is physiologically and functionally separate from the components of the circulatory immune system. Mucosal S-IgA responses are predominantly generated by the common mucosal immune system (CMIS) [Mestecky, J. Clin. Immunol. (1987), 7:265-276], in which immunogens are taken up by specialized lymphoepithelial structures collectively referred to as mucosa associated lymphoid tissue (MALT). The term common mucosal immune system refers to the fact that immunization at any mucosal site can elicit an immune response at all other mucosal sites. Thus, immunization in the gut can elicit mucosal immunity in the upper airways and vice versa.
Further, it is important to note that oral immunization can induce an antigen-specific IgG response in the systemic compartment in addition to mucosal IgA antibodies [McGhee, J. R. et al., (1993), Infect. Agents and Disease 2:55-73].
Most soluble and non-replicating antigens are poor mucosal immunogens, especially by the peroral route, probably because digestive enzymes degrade such antigens and such antigens have little or no tropism for the gut associated lymphoid tissue (GALT). Thus, a method for producing effective mucosal immunogens, and vaccines and immunogenic compositions containing them, would be desirable.
Native protein antigens such as PsaA, or immunogenic fragments thereof, stimulate an immune response when administered to a host. Recombinant proteins are promising vaccine or immunogenic composition candidates because they can be produced at high yield and purity and manipulated to maximize desirable activities and minimize undesirable ones. However, because they can be poorly immunogenic, methods to enhance the immune response to recombinant proteins are important in the development of vaccines or immunogenic compositions. Such antigens, especially when recombinantly produced, may elicit a stronger response when administered in conjunction with an adjuvant. An adjuvant is a substance that enhances the immunogenicity of an antigen. Adjuvants may act by retaining the antigen locally near the site of administration to produce a depot effect, facilitating a slow, sustained release of antigen to cells of the immune system. Adjuvants can also attract cells of the immune system, and may attract immune cells to an antigen depot and stimulate such cells to elicit an immune response.
Immunostimulating agents or adjuvants have been used for many years to improve the host immune response to, for example, vaccines. Intrinsic adjuvants, such as lipopolysaccharides, normally are components of the killed or attenuated bacteria used as vaccines. Extrinsic adjuvants are immunomodulators that are typically non-covalently linked to antigens and are formulated to enhance the host immune response. Aluminum hydroxide and aluminum phosphate (collectively commonly referred to as alum) are routinely used as adjuvants in human and veterinary vaccines. Currently, alum is the only adjuvant licensed for human use, although hundreds of experimental adjuvants such as cholera toxin B are being tested. However, these adjuvants have deficiencies. For instance, while cholera toxin B is not toxic in the sense of causing cholera, there is general unease about administering a toxin associated with a disease as harmful as cholera, especially if there is even the most remote chance of minor impurity. Also, it is generally believed that, for cholera toxin B to function effectively as an adjuvant, there must be some cholera toxin activity.
Thus, it would be desirable to enhance the immunogenicity of antigens, by methods other than the use of an adjuvant, especially in monovalent preparations; and, in multivalent preparations, to have the ability to employ such a means for enhanced immunogenicity with an adjuvant, so as to obtain an even greater immunological response.
A very promising immune stimulator is the lipid moiety N-palmitoyl-S-(2RS)-2,3-bis-(palmitoyloxy) propyl cysteine, abbreviated Pam3Cys. This moiety is found at the amino terminus of the bacterial lipoproteins that are synthesized with a signal sequence that specifies lipid attachment and cleavage by signal peptidase II. Synthetic peptides that by themselves are not immunogenic induce a strong antibody response when covalently coupled to Pam3Cys [Bessler et al., Research Immunology (1992) 143:548-552].
In addition to an antibody response, one often needs to induce a cellular immune response, particularly cytotoxic T lymphocytes (CTLs). Pam3Cys-coupled synthetic peptides are extremely potent inducers of CTLs, but no one has yet reported CTL induction by large recombinant lipoproteins.
As described in WO 90/04411, an analysis of the DNA sequence for the B31 strain of B. burgdorferi shows that the OspA protein is encoded by an open reading frame of 819 nucleotides starting at position 151 of the DNA sequence and terminating at position 970 of the DNA sequence (see FIG. 1 therein).
The first sixteen amino acid residues of OspA constitute a hydrophobic signal sequence of OspA. The primary translation product of the full length B. burgdorferi gene contains a hydrophobic N-terminal signal sequence which is a substrate for the attachment of a diacyl glycerol to the sulfhydryl side chain of the adjacent cysteine residue. Following this attachment, cleavage by signal peptidase II and the attachment of a third fatty acid to the N-terminus occurs. The complete lipid moiety is termed Pam3Cys. It has been shown that lipidation of OspA is necessary for immunogenicity, since OspA lipoprotein with an N-terminal Pam3Cys moiety stimulates a strong antibody response, while OspA lacking the attached lipid does not induce any detectable antibodies [Erdile et al., Infect. Immun., (1993), 61:81-90].
Published international patent application WO 93/10238 describes the DNA sequence of the psaA gene of S. pneumoniae strain (type 6B) and the PsaA protein encoded thereby of 37 kDa molecular weight. This sequence reveals that PsaA is a lipoprotein that employs a signal sequence similar to that used for OspA. Based on the findings regarding OspA, one might expect that lipidation of recombinant PsaA would be useful to enhance its immunogenicity; but, as discussed below, the applicants experienced difficulties in obtaining detectable expression of recombinant PsaA.
U.S. Pat. No. 4,624,926 to Inouye relates to plasmid cloning vectors, including a DNA sequence coding for a desired polypeptide linked with one or more functional fragments derived from an outer membrane lipoprotein gene of a gram negative bacterium. The polypeptide expressed by the transformed E. coli host cells comprises the signal peptide of the lipoprotein, followed by the first eight amino acid residues of the lipoprotein, which in turn are followed by the amino acid sequence of the desired protein. The signal peptide may then be translocated naturally across the cytoplasmic membrane and the first eight amino acids of the lipoprotein may then be processed further and inserted into the outer membrane of the cell in a manner analogous to the normal insertion of the lipoprotein into the outer membrane. Immunogenicity of the expressed proteins was not demonstrated.
Published international patent application WO91/09952 describes plasmids for expressing lipidated proteins. Such plasmids involve a DNA sequence encoding a lipoprotein signal peptide linked to the codons for one of the β-turn tetrapeptides QANY or IEGR, which in turn is linked to the DNA sequence encoding the desired protein.
Again, immunogenicity of the expressed proteins was not demonstrated.